SESSION 10

Structure and function of RNA

Organized by Z. Szweykowska-Kuliñska

39th Meeting of thePolish Biochemical SocietyGdañsk 16–20 September 2003

407 Lecture

Diseases caused by mitochondrial RNA mutations

Ewa Bartnik

Zak³ad Genetyki, Uniwersytet Warszawski, ul. Pawiñskiego 5A, Warszawa

The human mitochondrial genome is a small circularDNA molecule encoding the synthesis of 13 proteins,22 tRNAs and 2 rRNAs. Perturbations in mitochon-drial function due to mutations in genes encoded inboth mitochondrial and nuclear DNA can lead to a num-ber of diseases, including blindness, deafness,neuromuscular disorders and diabetes. Changes in mi-

tochondrial function have been associated with aging,and somatic mutations in mitochondrial DNA can oc-cur during carcinogenesis. Numerous mutations havebeen found in genes encoding tRNA and rRNA. I willpresent data on these mutations, including our own re-sults on analyzing mutations involved in mitochondrialmyopaties and mutations found in tumours.

408 Lecture

Ribozymes of the hepatitis delta virus: new findings on catalytic properties of ribo-nucleic acids

Jerzy Ciesio³ka

Instytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ

Although the delta ribozymes have been studied forfifteen years the most important information concern-ing their structure and mechanism of catalysis were ob-tained very recently. The determination of the crystalstructure of the genomic ribozyme is, undoubtedly, oneof the most important achievements of ‘ribozymology’in recent years. Details of the X-ray structure havegreatly stimulated further studies on the folding ofthese ribozymes into functionally active molecules aswell as on the mechanism of RNA catalysis. The abilityof the delta ribozymes to carry out general acid-base ca-talysis by nucleotide side chains has been assumed intwo proposed mechanisms of self-cleavage. Subse-quently, a similar strategy has been suggested for theribosome and peptide bond formation. Generalacid-base catalysis by nucleotide side chains increases

the catalytic repertoire of RNA and suggests that simi-lar strategies could operate in other RNA-catalyzed re-actions.

Recently, we have used the in vitro selection approachto select for antigenomic ribozyme variants that are ac-tive in the presence of Mg(II). The following ribozyme re-gions were randomized: L3, J1/4 and J4/2, and after 10cycles of selection-amplification, cloning and sequencingof the enriched ribozyme pool, several functionalribozymes were identified. Characterization of their cat-alytic activity, also in the presence of other than Mg(II)divalent metal ions, revealed that some nucleotide sub-stitutions changed ionic preferences of selected vari-ants. Interestingly, in some variants the cytosine residueproposed to play a crucial role in the cleavage mecha-nism of the wild type ribozyme was mutated.

409 Lecture

New mRNA 5’-cap analogues as useful tools in translation studies in eukaryota

Edward Dar¿ynkiewicz1, Jacek Jemielity1, Janusz Stêpiñski1, Joanna ¯uberek1, Anna NiedŸwiecka1, MagdalenaLewdorowicz1, Dorota Haber1, Ryszard Stolarski1, Robert Rhoads2

1 — Zak³ad Biofizyki IFD, Uniwersytet Warszawski, ul. ¯wirki i Wigury 93, 02-089 Warszawa, 2 — Department of Biochemistryand Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, USA

Synthetic analogues of 5’-terminal cap of eukaryoticmRNA and snRNA have played an important role in elu-cidating such physiological processes as mRNA transla-tion, pre-mRNA splicing, intracellular transport ofmRNA and snRNAs, and mRNA turnover. Particularly

useful are RNAs capped with synthetic analogues, whichare produced by in vitro transcription of a DNA templateusing a bacteriophage RNA polymerase in the presenceof ribonucleoside triphosphates and a cap dinucleotidesuch as m7Gp3G. Unfortunately, due to the presence of

234 Session 10. Structure and function of RNA 2003

a 3’-OH on both the 7-methylguanosine and guanosinemoieties, up to half of the mRNAs contain caps incorpo-rated in the reverse orientation [Pasquinelli et al. (1995)RNA’ 1: 957–967]. Previously we designed and synthe-sized two “anti-reverse” cap analogues (ARCAs),m73’dGp3G and m27,3’OGp3G, that cannot be incorpo-rated in the reverse orientation because of lack of free3’-OH in 7-methylguanosine moiety [Stepinski et al.(2001) RNA, 7: 1486–1495]. In the present study wehave synthesized several new cap analogues modified inthe C2’ and C3’ positions of 7-methylguanosine and inthe number of phosphate residues, m27,2’OGp3G, m72’d-Gp3G, m72’dGp4G, m27,2’OGp4G, m27,3’OGp4G,

m7Gp5G, and m27,3’OGp5G. These were analysed forconformation in solution, binding affinity to eIF4E, inhi-bition of in vitro translation, and the ability to stimulatecap-dependent translation in vitro when incorporatedinto mRNA. The results indicate that modifications atC2’, like those at C3’, prevent reverse incorporation, thattetra- and pentaphosphate cap analogues bind eIF4Eand inhibit translation more strongly than theirtriphosphate counterparts, and that tetraphosphate (butnot pentaphosphate) ARCAs promote cap-dependenttranslation more effectively than any cap analoguetested to date.Supported by the State Committee for Scientific Research:

PBZ-KBN-059/T09/10.

410 Lecture

Splicing of U12-dependent introns in plants

Artur Jarmo³owski

Department of Gene Expression, Adam Mickiewicz University, ul. Miedzychodzka 5, 60-371 Poznañ

While several animal U12-dependent introns havebeen analyzed thoroughly, no significant study of U12splicing in plants has been made. We scanned theArabidopsis genome for U12 introns. Our computa-tional analysis revealed that only 0.06% of all Arabi-dopsis introns have features characteristic of U12 inter-vening sequences. The U12 introns were distributedamong 69 genes, all contain one U12-dependent intronexcept for two genes that have two U12 introns. Therewas no clear preference for the position of U12-type in-tervening sequences within the genes analyzed.

Three U12-dependent introns were subjected to de-tailed splicing analysis in planta and in tobacco proto-plasts. The introns were derived from three differentArabidopsis genes, which encode nuclear cap-bindingprotein (AtCBP20), glutathione synthetase (Atgsh2)and LUMINIDEPENDENS (AtLD). The introns stud-ied were spliced efficiently in their normal context

within plant tissues. However, in tobacco protoplasts,excision from zein pre-mRNA was generally poor.There were notable differences in efficiency of the U12introns splicing. While AtCBP20 and Atgsh2 U12 typeintrons were spliced only at 10% and 5% respectively,AtLD intron was spliced with 50% efficiency. Deletionof natural exonic fragments from the LD construct de-creased splicing of the U12 intron to 9%, suggesting thepresence of a putative exonic enhancer within theexons. We identified by deletion analysis a 40-nucleo-tide fragment in the upstream exon that stimulatessplicing of the U12 intron more than 5 times. Splicingof U12 dependent introns in protoplasts was not en-hanced by over expression of known plant SR proteinsand U-rich RNA binding protein UBP1. Our experi-ments suggest that RBP45, a member of the planthnRNP-like protein family, can be involved in regula-tion of U12-dependent splicing in plants.

411 Lecture

Pathways for nuclear mRNA degradation in yeast

Joanna Kufel1, Laura Milligan2, Claire Torchet2, Cecile Bousquet-Antonelli2, David Tollervey2

1 — Zak³ad Genetyki, Uniwersytet Warszawski, ul. Pawinskiego 5a, 02-106 Warszawa, 2 — ICMB, University of Edinburgh, EH93JR Edinburgh, United Kingdom

Control of mRNA stability is a key step in the regula-tion of gene expression. During the degradation of cy-toplasmic mRNA, deadenylation-dependent removalof the 5’-cap by the decapping complex Dcp1p/Dcp2pis activated by the cytoplasmic Lsm1-7p complex.

Pathways that degrade mRNA precursors in theyeast nucleus have also been identified and different

pathways appear to utilize the same RNA degradationmachinery. Unspliced pre-mRNAs are rapidly de-graded predominantly 3’-5’ by the nuclear exosome,whereas 5’-3’ degradation by the exonuclease Rat1pplays a minor role. The nuclear exosome also func-tions in the degradation of pre-mRNAs that havefailed to undergo correct 3’-end formation. However,

2003 39th Meeting of the Polish Biochemical Society 235

in this pathway there is no evidence for significant5’-3’ degradation.

5’-3’ degradation of nuclear pre-mRNAs also requirestheir decapping prior to exonuclease digestion. Cellslacking nuclear Lsm2-8 proteins or the decapping fac-tor Dcp1p stabilize pre-mRNA degradation intermedi-ates that would normally be subject to 5’-3’ degrada-tion. These intermediates remain 5’-capped, indicatingthat the nuclear Lsm2-8p complex normally targetsRNA substrates for decapping.

Mature HS, GAL and MET mRNAs that are trappedin the nucleus due to a block in mRNA export are alsostabilized and remain 5’-capped in lsm or dcp1 mu-tants. These nuclear-restricted mRNAs remain poly-

adenylated until their degradation, indicating that nu-clear mRNA degradation does not involve incrementaldeadenylation, which is a key feature of cytoplasmicturnover. Nuclear Lsm8p can be UV cross-linked to nu-clear-restricted poly(A)+ RNA. This indicates that theLsm2-8p complex interacts directly with mRNA sub-strates to activate decapping prior to 5’-3’ degradation.

Nuclear-restricted HS and MET mRNAs are also de-graded 3’-5’ by Rrp6p, an exonuclease that is specifi-cally associated with the nuclear form of the exosomecomplex but not by the core exosome.

These data show that degradation of different nuclearsubstrates has distinct features suggesting that theyare targeted by related but distinct turnover pathways.

412 Lecture

New types of RNA within the cell

Maciej Szymañski, Marek ¯ywicki, Miros³awa Barciszewska, Jan BarciszewskiInstytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ

Recent analyses of the human genome revealed thatonly about 1.5% is transcribed and translated into pro-tein. Current data indicate that a considerable fractionof genomic DNA transcripts is responsible for regula-tion of gene expression on both transcriptional andposttranscriptional level. Interestingly both types ofmechanisms involve non-protein coding RNAs.

Noncoding RNAs (ncRNAs) are stable and functionalRNA molecules without coding capacity. They can bebroadly divided into two major group: housekeepingncRNAs and regulatory ncRNAs. The RNAs belongingto the first group are necessary for primary cell func-tions. The second group can be divided into several sub-classes depending on the mechanisms of action. Regu-latory ncRNAs are able to affect expression of pro-tein-coding genes on both transcriptional and post-transcriptional levels. These RNAs act by multiplemechanisms such as RNA-RNA base pairing,RNA-protein interactions and intrinsic RNA activityand regulate diverse cellular functions, including chro-

matin structure modification, RNA processing, mRNAstability and translation. Their involvement in somekey genetic processes, like genomic imprinting or Xchromosome inactivation in higher eukaryotes revealstheir importance in cell metabolism. Noncoding RNAshave been also implicated in development and their de-fects can lead to various genetic disorders such asPrader-Willi syndrome, Beckwith-Wiedemann syn-drome or cancerogenesis.

The fact, that over 98% of human genome does not en-code proteins stimulates efforts for ncRNAs gene find-ing. In contrast to protein coding genes, extremely vari-able structural properties of this type of molecules in ad-dition to lack of open reading frames makes them veryhard targets for computational based genome search.Till now there is lack of efficient method of ncRNA genesprediction. The important role of noncoding transcriptsis however evident from the analysis of the mousetranscriptome, where approximately one third of totalcDNA clones does not show protein-coding potential.

413 Oral Presentation

Analysis of putative human RNA methyltransferase that introduces methyl group onthe first anticodon position in pre-tRNA-Leu(CAA).

Bartosz Brzezicha1, Marcin Schmidt2, Izabela Maka³owska3, Zofia Szweykowska-Kuliñska1

1 — Zak³ad Ekspresji Genów, Instytut Biologii Molekularnej i Biotechnologii, Uniwersytet im. Adama Mickiewicza,ul. Miêdzychodzka 5, 60-371 Poznañ, 2 — Katedra Biotechnologii i Mikrobiologii ¯ywnoœci, Akademia Rolnicza im. AugustaCieszkowskiego, ul. Wojska Polskiego 48, 60-627 Poznañ, 3 — Computational Genomics Center, The Huck Institute for LifeSciences, Pennsylvania State University, 506 Wartik Lab, University Park, 16802 Pennsylvania, USA

Yeast m5C34-tRNA-Leu-methyltransferase Trm4 methy-lates first anticodon position in pre-tRNA-Leu(CAA) inintron-dependent manner. It is known that the absence of theenzyme is not lethal for the cell but causes disturbances in

mRNA decoding. Human genes coding for tRNA-Leu(CAA)contain22–25nt long introns.Wehave shown, that in thenu-clear HeLa cell extract human pre-tRNA-Leu(CAA) is, as inthecaseof yeast,methylated in the intron-dependentmanner.

236 Session 10. Structure and function of RNA 2003

Full sequence of the methyltransferase gene in yeasthas been established. Searching for orthologous se-quences in human GenBank databases revealed thepresence of ESTs with high orthology to yeast enzymesequence. We aligned two putative mRNAs that differfrom each other by the presence of longer N-terminusin the protein after translation. Both have the same lo-cus on fifth human chromosome: 5p15.32.

Both cDNAs have been amplified using HumanHeLa Marathon-Ready cDNA (Clontech) as a tem-plate. After sequencing we observed point mutationsthat were probably incorporated by Taq Polymeraseduring PCR reaction. In longer cDNA some mutationswas responsible for changing encoded amino-acids.Mutations have been reversed by site-directed muta-genesis. Afterward, both cDNAs have been cloned intoprokaryotic expression vector pET-28a(+) and the

shorter one has been also cloned into yeast expressionvector p416 GALS. Overexpression of shorter proteinin E. coli is efficient, contrary to the longer one. Enzy-matic tests of shorter protein expressed in E. coli re-sulted in the lack of the methyltransferase activity.Expected activity has not been also detected in yeastextract after transformation of yeast line in which theendogenous methyltransferase gene has been deleted.Analogous experiments will be performed for testingactivity of longer protein.

We have established localization of both proteins inHeLa cells. The proteins have been expressed as a fu-sion to GFP. We observed localization using confocalmicroscopy. Both proteins localize in nucleus withclearly visible nucleolar localization. This observationis in accordance to previous data from which we knowthat posttranscriptional modifications of tRNA andrRNA take place mainly in the nucleolus.

414 Oral Presentation

Analysis of the posttranscriptional regulation of DNA polymerase beta expression inthe rat cells

Ewa Grzybowska, Anna Wilczyñska, El¿bieta Sarnowska, Janusz SiedleckiZak³ad Biologii Molekularnej, Centrum Onkologii — Instytut, ul. Roentgena 5, 02-781 Warszawa

DNA polymerase beta plays a key role in base excisionrepair (BER). Its overexpression leads to a mutatorphenotype, characteristic in certain cancers and itsknockout causes apoptotic death of neurons during em-bryonic development in mice. Two DNA polymerasebeta transcripts are expressed in rat cells and they dif-fer (due to alternative polyadenylation) only in thelength of their 3’ untranslated regions (3’UTRs). Thetranscript containing the shorter 3’UTR is expressed inmost tissues, with significantly higher levels in testis,while the one with the longer 3’UTR is expressedmostly in brain and lungs. Both 3’UTRs contain spe-cific motifs — putative protein binding sites — thatcould be responsible for DNA polymerase beta post-transcriptional regulation and tissue-specific expres-sion of the two transcripts.

Putative regulatory sequences of the DNA polymer-ase beta have been isolated and cloned in order to ana-

lyze their ability to bind specific proteins in the yeastthree-hybrid system and EMSA. EMSA performed withprotein fractions from rat cells, confirmed specific pro-tein binding to the chosen mRNA regions. Three-hybridanalysis of one of these regions has revealed the inter-action with HAX-1 protein, a potent anti-apoptotic fac-tor.

The effect of both 3’UTR sequences on transcript sta-bility and translatability has also been analyzed usingboth, luciferase and beta-globin reporter systems. Wehave established that the presence of short 3’UTRcauses an increase in luciferase expression, in contrastto the long 3’UTR, which exerts opposite effect. Theseresults show that 3’UTRs play an important role in reg-ulation of DNA polymerase beta expression and furtheridentification of specifically binding proteins will helpto elucidate the mechanisms behind it.

415 Oral Presentation

Efficiency of RNA polyadenylation by poly(A) polymerase in Escherichia coli dependson bacterial growth rate

Jacek Jasiecki, Grzegorz WêgrzynKatedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk

RNA polyadenylation occurs not only in eukaryotesbut also in bacteria. In prokaryotes, polyadenylatedRNA molecules are usually degraded more efficiently

than non-modified transcripts. Here, we demonstratethat two transcripts, which were shown previously to besubstrates for poly(A) polymerase I (PAP I), Escherichia

2003 39th Meeting of the Polish Biochemical Society 237

coli lpp mRNA and bacteriophage lambda oop RNA, arepolyadenylated more efficiently in slowly growing cellsthan in rapidly growing bacteria. Intracellular levels ofPAP I varied in inverse proportion to bacterial growthrate. Moreover, transcription from a promoter for thepcnB gene (encoding for PAP I) was shown to be more ef-ficient under conditions of low bacterial growth rates.

We conclude that efficiency of RNA polyadenylation inE. coli is higher in slowly growing bacteria due to moreefficient expression of the pcnB gene. This may allowregulation of stability of certain transcripts (those sub-jected to PAP I-dependent polyadenylation) in responseto various growth conditions.

416 Poster

Synthesis of nucleosides tri- and tetraphosphates resistant to hydrolysis

Jacek Jemielity, Marcin Ka³ek, Janusz Stêpiñski, Edward Dar¿ynkiewicz

Zak³ad Biofizyki IFD, Uniwersytet Warszawski, ul. ¯wirki i Wigury 93, 02-089 Warszawa

The series of nucleosides tri- and tetraphosphatescontaining methylene group instead of terminalpyrophosphoric bond has been synthesized. Such modi-fied compounds are resistant to either chemical and en-zymatic hydrolysis [1, 2]. These derivatives may be ap-plied in studies of decapping process and other bio-chemical researches. The method used to insert themethylene bridge to the phosphate chain consists incoupling an imidazole derivative NpnIm withbisphosphonate[3]. The elaborated synthetic methodenables connecting the substrates with high yields anddoes not require a usage of any protective groups, whatmakes it very useful in synthesis of unstable com-pounds like for instance modified derivatives of7-methylguanosisne. Preparation of analogue contain-

ing two CH2 groups is far more complicated. Currently,we are carrying out a synthesis of GpCH2pCH2pG.Crucial step in route to this compound is a condensa-tion of protected guanosine with bis(O-benzylphosphonomethyl)phosphonic acid benzyl ester, viaMitsunobu reaction[4].References:1. Thevenod F, Anderie I, Schulz I (1994) J Biol Chem, 269:

24410.2. Tobin T, Akera T, Lee CY, Brody TM (1974) Biochem

Biophys Acta, 345: 102.3. Jemielity J et al, RNA — in press.4. Klein E, Mons S, Valleix A, Mioskowski C, Lebeau L (2002)

J Org Chem, 67146.Supported by the State Committee for Scientific Research:

PBZ-KBN-059/T09/10.

417 Poster

HIV RT-mediated site-specific nonhomologous RNA recombination

Anna Kurzynska-Kokorniak, Paulina Jackowiak, Marek Figlerowicz

Instytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ

Our preliminary studies conducted in vivo on bromemosaic virus (BMV) and in vitro on human immunodefi-ciency virus (HIV) showed that local hybridization be-tween RNA molecules (local RNA-RNA duplex forma-tion) can induce site-specific nonhomologous recombi-nation if a double-stranded region is accompanied byshort homologous sequences. A specific localization ofthe latter causes the local RNA-RNA duplex to exist intwo different conformations. We hypothesized that theobserved conformational dynamism of recombiningmolecules is responsible for the precise transfer of theviral polymerase from one template (called RNA donor)to the other (called RNA acceptor).

To verify the above hypothesis we have created an invitro system enabling effective studies on the mecha-nism of nonhomologous site-specific RNA recombina-

tion. Using this system we have tested the recombina-tion activity of HIV-1 reverse transcriptase (HIV-1 RT ?p51/p66 heterodimer) and of the p66/p66 homodimer.To establish the role of RNA structure in the studiedprocess, we have constructed several RNA donor andRNA acceptor molecules sharing short homologous se-quences of various length (3, 5 and 10 nucleotides). Wehave also tried to determine the enzymes’ ability toswitch from the 5’ end of the donor template to the 3’end of the acceptor template. Moreover, we have testedHIV-1 RT and p66/p66 homodimer recombination ac-tivities in the system that offers optimal conditions forsite-specific template switching by reverse transcrip-tase.

Our results indicate that neither local hybridizationbetween template molecules nor the presence of short

homologous sequences, can alone induce site-specifictemplate switching by HIV-1 RT or p66/p66 homo-dimer. Only the simultaneous occurrence and the

proper localization of both motifs lead to recombinantformation.

418 Poster

Appearance of novel PSTVd variants upon coinfection with two non-infectiousPSTVd clones

Wojciech Podstolski, Anna Góra-Sochacka, W³odzimierz Zagórski-Ostoja

Instytut Biochemii i Biofizyki, Polska Akademia Nauk, ul. Pawiñskiego 5A, 02-106 Warszawa

In the course of studies on the genetic stability of theinfectious PSTVd variants I2 and I4, among a diversepopulation of progeny molecules, two non-infectiousclones, I2-50 and

I4-37, were found. The I2-50 and I4-37 variants areconsidered non-infectious on the basis of lack of visiblesymptoms on Lycopersicon esculentum ‘Rutgers’ testplants and negative dot blot hybridization results.

The presence of such mutants among the populationof viable variants remains a puzzling observation. Toinvestigate the biological properties and possible inter-actions between these molecules, two types of experi-ments were carried out.

Double inoculation experiments were performed inwhich monomeric full-length cDNA copies of the I2-50and I4-37 variants were used. None of the twenty plantsinoculated with either variant (I2-50 or I4-37) used aloneas controls, or a mixture of the variants showed visiblesymptoms. However, dot blot hybridization analyses re-vealed that 7 out of the 30 doubly (I2-50 and I4-37) inocu-lated plants were PSTVd positive. The PSTVd progenyisolated from three selected plants was analyzed reveal-ing the presence of 10 different PSTVd variants. Eight ofthem have not been previously described.

The same experiment was conducted, but usingmonomeric (+) RNA transcripts. Contrary to the re-sults obtained with the cDNA copies, all inoculationsyielded positive signals (in 3/15 plants for I2-50, 2/15for I4-37 and 10/30 for the mixture of transcripts). Thisresult shows that transcripts of apparently non-in-fectious cDNA can induce infection. Analysis of PSTVdclones isolated from three selected plants revealed thatall the progeny of I2-50 and the mixture of both tran-scripts reversed to the infectious I2 parental type. Incontrast in the progeny of I4-37, 8 sequences were de-tected, 6 of them were not previously described.

It must be stressed that in neither of these two typesof experiments, were I2-50 nor I4-37 sequences de-tected. Analysis and comparison of the sequences iden-tified in both experiments with sequences retrievedfrom the NCBI database made it possible to identifymutations responsible for the apparent lack ofinfectivity and draw general conclusions on nucleotidepreferences at certain positions in the PSTVd genome.Our experiments show that mixed infection with twoapparently non-infectious PSTVd variants is followedby the appearance of progeny recombinants with re-stored regions allowing systemic infection.

419 Poster

AtCBP20 — the cap-binding protein from Arabidopsis thaliana has an nuclear local-ization signal

Artur Rogowski, Maciej Kmieciak, Artur Jarmo³owski

Department of Gene Expression, Adam Mickiewicz University, ul. Miedzychodzka 5, 60-371 Poznañ

All transcripts made by eukaryotic RNA polymeraseII are co-transcriptionally modified at their 5’ ends bythe addition of a cap structure. It has been shown thatthe cap structure is involved in several different as-pects of RNA metabolism, such as pre-mRNA splicing,RNA export from the nucleus to the cytoplasm andpolyadenylation. These functions are mediated by a nu-clear complex of cap-binding proteins called CBC. Itconsists of two cap-binding proteins: CBP20 and

CBP80. We have recently identified and characterizeda similar cap-binding complex from A. thaliana.AtCBP20 has a predicted molecular mass of 30 kDa.This protein contains a highly conserved canonicalRNA-binding domain and a plant specific longC-terminal tail. The C-terminal part of AtCBP20 is richin glycine and arginine residues. It has been shown thatCBC from HeLa cells is transported into the nucleusdue to nuclear localization signal (NLS) located in the

2003 39th Meeting of the Polish Biochemical Society 239

N-terminal domain of CBP80. Human CBP20 does notcontain any NLS. Using transient expression intransfected tobacco protoplasts and fluorescent micros-copy, we have shown that CBP20 from A. thaliana hasits own nuclear localization signal. We have recentlyshown that this signal consist of two separated ele-

ments. Mutations in this motifs have revealed that theyfunction in nuclear transport independently. Our re-sults indicate that this NLS is located in the plant spe-cific C-terminal tail of AtCBP20. This data suggest thatin plants CBP20 is responsible for transport of thecap-binding complex from the cytoplasm to the nucleus.

420 Poster

Studies of SIAH1, potential mRNA target for NANOS1-PUMILIO2 protein complexin the human germ cells

Anna Spik1, S³awomir Oczkowski2, Jacek B³a¿ewicz3, Maciej Kotecki1, Piotr Formanowicz3, JadwigaJaruzelska1

1 — Polska Akademia Nauk, Instytut Genetyki Cz³owieka, ul. Strzeszyñska 32, 60-479 Poznañ, 2 — Instytut Informatyki,Politechnika Poznañska, ul. Piotrowo 3A, Poznañ, 3 — Polska Akademia Nauk, Instytut Chemii Bioorganicznej,ul. Noskowskiego 12, Poznañ

The Nanos-Pumilio complex is Drosophila trans-lational repressor in morphogenesis, and in develop-ment of the germ cells. We have recently identified ho-mologous protein complex (NANOS1-PUMILIO2) inthe male human germ-line. The high conservation ofthe RNA-binding domains of these proteins suggeststhat they may function as translational regulators alsoin humans. Therefore, identification of target mRNAsseems crucial to understand the role of NANOS1 andPUMILIO2 in development of the human germ-line.

Electronic searches of the GeneBank using softwarewe have generated enabled us to retrieve candidate hu-man mRNA targets of the NANOS1-PUMILIO2 com-plex. These candidate mRNAs contain 3’UTR motifsidentical to motifs of the known mRNA targets of

Nanos and Pumilio in Drosophila. We found that one ofthese candidates is SIAH1. This gene is homologous toSina, the fertility gene of Drosophila. Indeed, our exper-iments demonstrated that SIAH1 is expressed in hu-man testes. The bend-shift approach has shown thatNANOS1 and PUMILIO2 bind 3’UTR of the SIAH1mRNA. In the next step, the conserved 3’UTR motifs ofSIAH1 were mutated by introducing nucleotide substi-tutions using site directed mutagenesis. The binding ef-ficiency of NANOS1-PUMILIO2 complex to 3’UTR mu-tated variants was substantially diminished. Althoughfurther tests are needed we believe that SIAH1 isstrong candidate for the NANOS1-PUMILIO2 complexmRNA target in the human germ cells.

421 Poster

Study of cleavage of the phosphodiester bond of mRNA 5’-cap analogs promoted bycopper ion and copper complexes

Alicja Stachelska1, Satu Mikkola2, Zbigniew Wieczorek1, Sanna Anderson2, Monika Pietrzak1, Satu Valakoski2

1 — Department of Physics and Biophysics, University of Warmia and Mazury in Olsztyn, ul. Oczapowskiego 4, 10-719 Olsztyn,2 — Department of Chemistry, University of Turku, FIN-20014 Turku, Finland

The aim of this studies was to investigate the hydroly-sis of mRNA compound ApppA and 5’-cap analogs:m7GpppG and m7GppppG in the presence of Cu2+ ionsand of Cu2+TerPy and Cu2+BiPy complexes to find effi-cient catalysts for cleavage its phosphodiester bound.

Comparison of rate constants of hydrolysis of ApppA,m7GpppG in the presence and absence of Cu2+ ionshows its significant role in the cleavage of thephospodiester bond of studied compounds. The resultsshow that hydroxoligand of metal ion catalyst is in-volved in this reaction. Comparison between the hydro-

lysis of ApppA, m7GpppG and m7GppppG in the pres-ence of Cu2+ complexes show that the complex ofCu2+TerPy is more effective catalyst than Cu2+BiPy forhydrolysis of the cap-analogs at pH 8.0. The hydrolysisof ApppA in presence Cu2+TerPy proceeds about 2.7times faster than in the presence of Cu2+BiPy. How-ever, m7GpppG hydrolysis in the presence ofCu2+TerPy is about 1.33 times faster than that in thepresence of Cu2+BiPy. The hydrolysis of m7GppppG isabout one order of magnitude faster in the presence ofCu2+TerPy than in the presence of Cu2+BiPy.

240 Session 10. Structure and function of RNA 2003

The results support suggestion that a hydroxoligandof a phosphate-bound metal ion acts as a nucleophile inthe hydrolysis reaction [2]. The role of the Cu2+ com-plex catalysts is significant, but is more complicatedthan for Cu2+ ion promoted hydrolysis [1]. The acidicaquoligand may be needed for the phospodiester bond

cleavage, more than one process. In some cases suffi-cient catalysis can be induced be one metal ion, butsometimes two separate catalysts are required.References:1. Anderson S, LDhde M, Mikkola S, Morris G, Stachelska A,

Valakoski S, Williams NH (2002) Collect Symp, Ser 5.2. Valakoski S, Heiskanen S, Anderson S, LDhde M, Mikkola

S (2002) J Chem Soc, Perkin Trans, 2604–610.

422 Poster

Homologous RNA recombination as a factor stabilizing and destabilizing a viral genome

Anna Urbanowicz1, Magdalena Alejska1, Marek Figlerowicz1, Jozef Bujarski2

1 — Instytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ, 2 — Plant MolecularBiology Center and Biology Department, Northern Illinois University, DeKalb, USA

Enormous genetic variability is one of the most un-usual features of RNA viruses. There are two mainsources of the genetic polymorphism of RNA genomes:error prone replication and RNA recombination.

Error prone replication introduces into a viral ge-nome a wide spectrum of point mutations. They canarise so frequently because RNA polymerases lackproofreading activity. The estimated average mutationrate amounts to 10–4 per nucleotide per replication cy-cle. RNA recombination is responsible for more pro-found changes within the viral genome like sequencedeletion, sequence insertion or the exchange of the en-tire coding domain. In the course of our studies we aretrying to establish what the real role of homologous re-combination in the virus life cycle is and this way tolearn more about the mechanisms generating new viralstrains or species. The object of our study is brome mo-saic virus (BMV) — a model (+)RNA virus of plants. Itsgenome consists of three RNA segments, RNA 1, 2 and3. To determine the frequency of homologous cross-

overs between the same genomic BMV molecules, weinduced mixed infections in plants, using pairs of BMVgenomic RNAs bearing marker mutations in codingand noncoding regions.

We observed that marker mutations in 3’ and 5’noncoding regions of BMV RNAs were very efficientlyremoved by imprecise replication. An analysis of thedistribution of the stable marker mutations in the na-scent virus revealed that homologous recombinationevents occur very often during virus genome replica-tion. The frequency of recombination in the coding re-gions ranged from 10% to 25%. We also identified a re-combination hot spot in the intergenic region of RNA3,where the frequency of crossovers reached 71%. Thatregion might serve as a place for the exchanging ofwhole genes. In general our results suggest that the ma-jority of nascent viruses are recombinants. This indi-cates that homologous RNA recombination is one of themain factors responsible for both the stabilization anddestabilization of the viral genome.

423 Poster

A procedure to generate in vitro RNA transcripts with homogenous 3’ ends

Agnieszka Wich³acz, Jerzy Ciesio³ka

Instytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ

It is known that during in vitro run–off transcriptionRNA polymerases often generate heterogeneous 3’ends. In particular, T7 RNA polymerase attaches addi-tional nucleotides to the nascent RNA transcript and atthe same time may show premature termination closeto its 3’ end. This results in decreased number of thefull-length, correctly transcribed products. However, inmany applications such as NMR, crystallization studies

and ligation reactions homogeneous transcripts are in-dispensable.

One approach for the preparation of RNA transcriptswith homogeneous 3’ terminus is the use of ribozymeswhich are attached to their 3’ ends. These constructscan cleave-off themselves acting in cis. On the contraryto hammerhead or hairpin ribozymes, delta ribozymesdo not have particular requirements on the sequence

2003 39th Meeting of the Polish Biochemical Society 241

upstream of the cleavage site and then can be applied toany RNA. In this communication we have shown thatthe antigenomic delta ribozyme could also be applied intrans to correctly process the 3’ ends of RNA tran-scripts. Two different transcripts were extended withthe sequence of only 7 nucleotides complementary to

the ribozyme recognition site. Following the cleavagereaction with the ribozyme acting as ‘molecular scis-sors’ homogeneity of transcripts was proven by TLCanalysis of their 3’ terminal nucleotides. The proposedprocedure is useful for virtually any RNA molecule thatwe desire to be homogeneous at its 3’ end.

242 Session 10. Structure and function of RNA 2003